Lifetimes and Reactivities of Some 1,2-Didehydroazepines Commonly Used in Photoaffinity Labeling Experiments in Aqueous Solutions

Abstract

The reactive 1,2-didehydroazepine (cyclic ketenimine) intermediates produced upon photolysis of phenyl azide, 3-hydroxyphenyl azide, 3-methoxyphenyl azide, and 3-nitrophenyl azide in water and in HEPES buffer were studied by laser flash photolysis techniques with UV-vis detection of the transient intermediates. The lifetimes of the 1,2-didehydroazepines were obtained along with the absolute rate constants of their reactions with typical amino acids, nucleosides, and other simple reagents present in a biochemical milieu. The nitro substituent greatly accelerates the bimolecular reactions of the cyclic ketenimines, and the 3-methoxy group greatly decelerates the absolute reactivity of 1,2-didehydroazepines. The intermediate produced by photolysis of 3-hydroxyphenyl azide is much more reactive than the intermediate produced by photolysis of 3-methoxyphenyl azide. We propose that the hydroxyl-substituted 1,2-didehydoazepines rapidly (<10 micros) tautomerize in water to form azepinones and much more rapidly than the corresponding 3-methoxy-substituted cyclic ketenimines undergo hydrolysis. Azepinones react more rapidly with nucleophiles than do methoxy-substituted 1,2-didehydroazepines and are the active species present upon the photolysis of 3-hydroxyphenyl azide in aqueous solution.

Molecular Determinants of Matrix Metalloproteinase-12 Covalent Modification by a Photoaffinity Probe

“…Triplet nitrene may covalently modify the ⑀ amino group of a lysine to form the structure reported in Scheme 2 (adduct 2b). Simple phenylazides produce dedihydroazepine upon photolysis in water or buffer (39). However, the photogenerated intermediate was also reliant on the nature of the substituents of the phenyl ring (36 -38).…”

Proximity-accelerated Chemical Coupling Reaction in the Benzodiazepine-binding Site of γ-Aminobutyric Acid Type A Receptors

“…Photoaffinity labeling represents a method for investigation of ligand-binding sites on unmodified receptors provided the lifetime of the photo-generated intermediate is very short (40). Aromatic azides photochemically generate nitrenes, which are subjected to rearrangements to cyclic ketenimines, the reactivity of which is highly dependent on the aromatic ring substitutions (17). Photoaffinity labeling experiments using Ro15-4513 might generate long-lived intermediates, leading to selection of more reactive amino acid residues (e.g. )…”

“…However, these results should take into account possible formation of long-lived reactive intermediates after photoactivation (16,17). An alternative approach studied consequences of single point mutation in GABA A receptors using radioactive ligand binding assay and/or electrophysiological measurements; by this, a large number of amino acid residues were identified on both ␣ and ␥ subunits to build up the benzodiazepine-binding site (18,19).…”

Computational Study of the Curtius-like Rearrangements of Phosphoryl, Phosphinyl, and Phosphinoyl Azides and Their Corresponding Nitrenes

“…Qualitatively, the endothermic of the Curtius-like rearrangement of 32, as compared to exoergic rearrangement of 30, can be rationalized by considering the relative bond strengths of the products. The experimental bond dissociation energies (BDE) of a O 2 N-OCH 3 The observed products of the photolysis of diphenoxyphosphoryl azide contained no phosphonamidate arising from a Curtius-like rearrangement and were exclusively C-H insertion products derived from the nitrene. 4 There are also no known experimental examples of a phosphoryl azide yielding Curtius-like rearrangement products.…”

“…2 It was generally believed that the photolysis of aryl azides produce arylnitrenes that indiscriminately react with nearby residues and organic substrates to form robust linkages, but it was later shown that the reactive species is a ketenimine, which is more discriminate in its reactivity, and hence less desirable as a photoaffinity labeling reagent. 3 We are very interested in developing indiscriminate, reactive nitrenes from photoactivatable precursors, especially ones which are suitable for photoaffinity labeling. In this vein, we considered phosphinoyl-, phosphinyl-, and phosphorylnitrenes as possible photoaffinity agents, with each of these possible nitrenes being derived from the appropriately substituted azides.…”